U.S. patent application number 16/844414 was filed with the patent office on 2021-03-04 for method for preventing and/or treating aging-associated cognitive impairment and neuroinflammation.
This patent application is currently assigned to THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY. The applicant listed for this patent is THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY. Invention is credited to Edgar G. ENGLEMAN, Khoa Dinh NGUYEN.
Application Number | 20210059985 16/844414 |
Document ID | / |
Family ID | 1000005209911 |
Filed Date | 2021-03-04 |
United States Patent
Application |
20210059985 |
Kind Code |
A1 |
NGUYEN; Khoa Dinh ; et
al. |
March 4, 2021 |
METHOD FOR PREVENTING AND/OR TREATING AGING-ASSOCIATED COGNITIVE
IMPAIRMENT AND NEUROINFLAMMATION
Abstract
The present invention is directed to a method for preventing
and/or treating aging-related cognitive impairment in the central
nervous system. The method comprises administering to a subject in
need thereof a Ppargc1a activator
2-(4-tert-butylphenyl)-1H-benzimidazole,
2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole, in an effective
amount. A preferred route of administration is oral
administration.
Inventors: |
NGUYEN; Khoa Dinh; (San
Francisco, CA) ; ENGLEMAN; Edgar G.; (Atherton,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR
UNIVERSITY |
Stanford |
CA |
US |
|
|
Assignee: |
THE BOARD OF TRUSTEES OF THE LELAND
STANFORD JUNIOR UNIVERSITY
Stanford
CA
|
Family ID: |
1000005209911 |
Appl. No.: |
16/844414 |
Filed: |
April 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15781756 |
Jun 6, 2018 |
10653669 |
|
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PCT/US2016/065972 |
Dec 9, 2016 |
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16844414 |
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62267437 |
Dec 15, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 235/18 20130101;
A61K 31/4184 20130101; A61K 9/0053 20130101; A61K 31/404 20130101;
A61P 25/00 20180101; A61P 29/00 20180101 |
International
Class: |
A61K 31/404 20060101
A61K031/404; A61K 31/4184 20060101 A61K031/4184; C07D 235/18
20060101 C07D235/18; A61P 25/00 20060101 A61P025/00; A61P 29/00
20060101 A61P029/00; A61K 9/00 20060101 A61K009/00 |
Claims
1. A method of suppressing neuroinflammation or systemic
inflammation and metabolic abnormalities of microglia in the brain
comprising the step of: administering to a subject in need thereof
an effective mount of a compound
2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole, or a
pharmaceutically acceptable salt thereof.
2. The method according to claim 1, wherein said method reverses or
alleviates cognitive behavioral dysfunction of the subject.
3. The method according to claim 1, wherein said method suppresses
neuroinflammation and/or systemic inflammation of the subject.
4. The method according to claim 1, wherein said method suppresses
metabolic abnormalities of microglia in the brain of the
subject.
5. The method according to claim 1, wherein said compound is
administered by systemic administration.
6. The method according to claim 1, wherein said compound is
administered by oral administration.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for preventing
and/or treating aging-associated cognitive impairment and
neuroinflammation by administering to a subject a Ppargc1a
activator 2-(4-tert-butylphenyl)-1H-benzimidazole,
2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole.
BACKGROUND
[0002] Microglia are immune cells that are located only in the CNS.
Microglia originate from a yolk-sac hematopoietic progenitor, which
populates the brain during embryogenesis (Ginhoux et al Science
2009). During homeostatic conditions, microglia carry out
reparative processes such as debris clearance. They also produce an
arsenal of inflammatory mediators, which could be released upon
receiving pathological stimuli, to initiate and sustain
neuroinflammation. Similar to other immune cells, microglial
activation is a bio-energetically demanding process. What currently
remains elusive is how microglial metabolism becomes maladaptive
and contributes to the inflammatory transformation of these
cells.
[0003] Inflammatory responses in the brain, which can be
demonstrated by the presence of pro-inflammatory molecules and
changes in the properties of microglia, are a common feature of
human neurodegenerative diseases (Alzheimer's Res Ther., 7(1):56.
doi: 10.1186/s3195-015-0139-9, 2015). Yong (The Neuroscientist,
16:408-420, 2010) reports that inflammation of the central nervous
system (CNS) (neuroinflammation) is a feature of all neurological
disorders, and microglia activation results in elevation of many
inflammatory mediators within the CNS.
[0004] Aging is associated with a progressive loss of tissue
function, resulting in an increased susceptibility to aging-related
disorders. A consequence of physiological aging is a greater
susceptibility to memory impairment following an immune challenge
such as infection, surgery, or traumatic brain injury. The
neuroinflammatory response, produced by these challenges, results
in increased and sustained production of pro-inflammatory cytokines
in the otherwise healthy aging brain. Sensitized microglia are a
primary source of this exaggerated neuroinflammatory response and
appear to be a hallmark of the aging brain. The causes and effects
of aging-induced microglial sensitization include dysregulation of
the neuroendocrine system, potentiation of neuroinflammatory
responses following an immune challenge, and the impairment of
memory (Barrientos et al, Neuroscience 309:84-99, 2015). Aging is
associated with a decline in cognitive performance, and is the
biggest risk factor for the development of Alzheimer's disease
(AD). Mosher et al (Biochem Pharmacol 88:594-604, 2014) report
microglial dysfunction in brain aging and Alzheimer's disease.
Nevertheless, the role of intrinsic regulatory pathways in
microglia in these phenomena remains unexplored.
[0005] Since the population of aging individuals is rapidly
expanding and neuroinflammation is a pro-longed process that
develops during mid-life (40-60 years old) and accelerates with old
age (over 60 years old), it is important to identify a novel
therapeutic target for treating as well as preventing aging-related
disorders. There is a need for a therapy to inhibit
microglia-mediated neuroinflammation and its pathological
consequences in aging.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] In FIGS. 1-4, Veh=0.5% methylcellulose oral gavage,
ZLN=ZLN005 in Veh.
[0007] FIG. 1 shows % of microglia that either express glucose
transporter Slc2a1 or have taken up glucose fluorescent analog
2-NBDG in young animal, vehicle-treated older animals, or
ZLN-treated older animals; n=6 animals per condition.
[0008] FIG. 2 shows % of microglia that express CCL2 or TNF-.alpha.
in young animals, vehicle-treated older animals, or ZLN-treated
older animals; n=6 animals per condition.
[0009] FIG. 3 shows the concentration of TNF-.alpha. in serum of
young animals, vehicle-treated older animals, or ZLN-treated older
animals; n=6 animals per condition.
[0010] FIG. 4 shows the marble burying score in young animal,
vehicle-treated older animals, or ZLN-treated older animals; n=8-9
animals per condition.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The inventors have discovered that Ppargc1a, a pleotropic
regulator of cellular metabolism in many cell types including
microglia, is an important regulator of aging-associated
neuropathic disorders. The inventors have discovered a connection
between Ppargc1 activation in microglia and its effect on the
cognitive and motor functions of the whole organism. The inventors
have discovered that activating Ppargc1a with compounds such as
ZLN005 ameliorates microglial dysfunction and improves cognitive
performance in older animals.
[0012] The present invention is directed to a method for preventing
or treating age-associated cognitive impairment or
neuroinflammation. The method comprises the step of administering a
Ppargc1a activator to a subject in need thereof, in an amount
effective to prevent, arrest, or reverse the development of
aging-related symptoms. "Preventing," as used herein, refers to
arresting or slowing progression of age-associated cognitive
impairment or neuroinflammation. "Treating," as used herein, refers
to reverse, alleviate, or reduce age-associated cognitive
dysfunction or neuroinflammation. The subject is an aging subject
(45-60 years old) or an old subject (over 60 years old).
[0013] 2-(4-tert-Butylphenyl)-1H-benzimidazole,
2-[4-(1,1-Dimethylethyl)phenyl]-1H-benzimidazole, CAS Number
49671-76-3, also known as ZLN005, is an effective Ppargc1a
activator useful for treating aging. The chemical structure of
ZLN005 is shown below.
##STR00001##
[0014] The inventors have demonstrated that microglia in older mice
are more glycolytic, evidenced by their increased utilization of
glucose as its energy substrate and an upregulation of the glucose
transporter Slc2a1. Treating these older animals with ZLN005 led to
a significant inhibition of glucose uptake as well as Slc2a1
expression in microglia.
[0015] The inventors have shown that microglia in older mice
exhibit an inflammatory phenotype, evidenced by a significant
increase in CCL2 and TNF-.alpha. production. Tumor necrosis factor
(TNF or TNF.alpha.) is a cell signaling protein (cytokine) involved
in local and systemic inflammation and is one of the cytokines that
make up an acute phase reaction. The chemokine (C--C motif) ligand
2 (CCL2) is a small cytokine, which recruits monocytes, memory T
cells, and dendritic cells to the sites of inflammation produced by
either tissue injury or infection. By administering ZLN005 to older
animals, CCL2 and TNF-.alpha. production in microglia decreased and
neuroinflammation was suppressed. In addition, ZLN005 treatment
suppressed systemic inflammation in older mice, evidenced by its
inhibitory effect on serum TNF-.alpha. level.
[0016] The inventors have provided evidence that treatment with
ZLN005 reduced or reversed behavioral dysfunction, i.e., cognitive
dysfunction, in older mice in a marble-burying test, which is a
functional assay to assess cognitive competency.
Pharmaceutical Compositions
[0017] The present invention provides pharmaceutical compositions
comprising one or more pharmaceutically acceptable carriers and an
active compound of 2-(4-tert-butylphenyl)-1H-benzimidazole,
2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole (ZLN005), or a
pharmaceutically acceptable salt, or a solvate thereof. The active
compound or its pharmaceutically acceptable salt or solvate in the
pharmaceutical compositions in general is in an amount of about
0.01-20% (w/w) for a topical formulation; about 0.1-5% for an
injectable formulation, 0.1-5% for a patch formulation, about 1-90%
for a tablet formulation, and 1-100% for a capsule formulation.
[0018] In one embodiment, the pharmaceutical composition can be in
a dosage form such as tablets, capsules, granules, fine granules,
powders, syrups, suppositories, injectable solutions, patches, or
the like. In another embodiment, the pharmaceutical composition can
be an aerosol suspension of respirable particles comprising the
active compound, which the subject inhales. The respirable
particles can be liquid or solid, with a particle size sufficiently
small to pass through the mouth and larynx upon inhalation. In
general, particles having a size of about 1 to 10 microns,
preferably 1-5 microns, are considered respirable.
[0019] In another embodiment, the active compound is incorporated
into any acceptable carrier, including creams, gels, lotions or
other types of suspensions that can stabilize the active compound
and deliver it to the affected area by topical applications. The
above pharmaceutical composition can be prepared by conventional
methods.
[0020] Pharmaceutically acceptable carriers, which are inactive
ingredients, can be selected by those skilled in the art using
conventional criteria. Pharmaceutically acceptable carriers
include, but are not limited to, non-aqueous based solutions,
suspensions, emulsions, microemulsions, micellar solutions, gels,
and ointments. The pharmaceutically acceptable carriers may also
contain ingredients that include, but are not limited to, saline
and aqueous electrolyte solutions; ionic and nonionic osmotic
agents such as sodium chloride, potassium chloride, glycerol, and
dextrose; pH adjusters and buffers such as salts of hydroxide,
phosphate, citrate, acetate, borate; and trolamine; antioxidants
such as salts, acids and/or bases of bisulfite, sulfite,
metabisulfite, thiosulfite, ascorbic acid, acetyl cysteine,
cysteine, glutathione, butylated hydroxyanisole, butylated
hydroxytoluene, tocopherols, and ascorbyl palmitate; surfactants
such as lecithin, phospholipids, including but not limited to
phosphatidylcholine, phosphatidylethanolamine and phosphatidyl
inositiol; poloxamers and poloxamines, polysorbates such as
polysorbate 80, polysorbate 60, and polysorbate 20, polyethers such
as polyethylene glycols and polypropylene glycols; polyvinyls such
as polyvinyl alcohol and povidone; cellulose derivatives such as
methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose,
carboxymethyl cellulose and hydroxypropyl methylcellulose and their
salts; petroleum derivatives such as mineral oil and white
petrolatum; fats such as lanolin, peanut oil, palm oil, soybean
oil; mono-, di-, and triglycerides; polymers of acrylic acid such
as carboxypolymethylene gel, and hydrophobically modified
cross-linked acrylate copolymer; polysaccharides such as dextrans
and glycosaminoglycans such as sodium hyaluronate. Such
pharmaceutically acceptable carriers may be preserved against
bacterial contamination using well-known preservatives, these
include, but are not limited to, benzalkonium chloride,
ethylenediaminetetraacetic acid and its salts, benzethonium
chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal,
and phenylethyl alcohol, or may be formulated as a non-preserved
formulation for either single or multiple use.
[0021] For example, a tablet formulation or a capsule formulation
of the active compound may contain other excipients that have no
bioactivity and no reaction with the active compound. Excipients of
a tablet or a capsule may include fillers, binders, lubricants and
glidants, disintegrators, wetting agents, and release rate
modifiers. Binders promote the adhesion of particles of the
formulation and are important for a tablet formulation. Examples of
excipients of a tablet or a capsule include, but not limited to,
carboxymethylcellulose, cellulose, ethylcellulose,
hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch,
tragacanth gum, gelatin, magnesium stearate, titanium dioxide,
poly(acrylic acid), and polyvinylpyrrolidone. For example, a tablet
formulation may contain inactive ingredients such as colloidal
silicon dioxide, crospovidone, hypromellose, magnesium stearate,
microcrystalline cellulose, polyethylene glycol, sodium starch
glycolate, and/or titanium dioxide. A capsule formulation may
contain inactive ingredients such as gelatin, magnesium stearate,
and/or titanium dioxide.
[0022] For example, a patch formulation of the active compound may
comprise some inactive ingredients such as 1,3-butylene glycol,
dihydroxyaluminum aminoacetate, disodium edetate, D-sorbitol,
gelatin, kaolin, methylparaben, polysorbate 80, povidone, propylene
glycol, propylparaben, sodium carboxymethylcellulose, sodium
polyacrylate, tartaric acid, titanium dioxide, and purified water.
A patch formulation may also contain skin permeability enhancer
such as lactate esters (e.g., lauryl lactate) or diethylene glycol
monoethyl ether.
[0023] Topical formulations including the active compound can be in
a form of gel, cream, lotion, liquid, emulsion, ointment, spray,
solution, and suspension. The inactive ingredients in the topical
formulations for example include, but not limited to, diethylene
glycol monoethyl ether (emollient/permeation enhancer), DMSO
(solubility enhancer), silicone elastomer (rheology/texture
modifier), caprylic/capric triglyceride, (emollient), octisalate,
(emollient/UV filter), silicone fluid (emollient/diluent), squalene
(emollient), sunflower oil (emollient), and silicone dioxide
(thickening agent).
METHOD OF USE
[0024] The present invention is directed to a method of preventing
and/or treating aging-related cognitive impairment and/or
neuroinflammation. The method prevents aging-related cognitive
impairment and neuroinflammation of the central nervous system
and/or reduces or reverses these symptoms once developed. The
method comprises the steps of first identifying a subject in need
thereof, and administering to the subject the active compound of
ZLN005, in an amount effective to treat aging-related symptoms. "An
effective amount," as used herein, is an amount effective to treat
an aging-related condition by reducing its symptoms.
[0025] In one embodiment, the method reverses or reduces behavioral
dysfunctions in an older or aging patient. In one embodiment, the
method suppresses neuroinflammation as well as systemic
inflammation of the subject. In one embodiment, the method
suppresses metabolic abnormalities of microglia in the brain of the
subject.
[0026] The pharmaceutical composition of the present invention can
be applied by local administration and systemic administration.
Local administration includes topical administration. Systemic
administration includes, but not limited to oral, parenteral (such
as intravenous, intramuscular, subcutaneous or rectal), and inhaled
administration. By systemic administration, the active compound
first reaches plasma and then distributes into target tissues. Oral
administration is a preferred route of administration for the
present invention.
[0027] Dosing of the composition can vary based on the extent of
the injury and each patient's individual response. For systemic
administration, plasma concentrations of the active compound
delivered can vary; but are generally 1.times.10-10-1.times.10-4
moles/liter, and preferably 1.times.10-8-1.times.10-5
moles/liter.
[0028] In one embodiment, the pharmaceutical composition is
administrated orally to the subject. The dosage for oral
administration is generally 1-100, and preferably 1-50, or 1-25
mg/kg/day. For example, the active compound can be applied orally
to an adult human at 50-1000 mg/dosage, or 100-600 mg/dosage, 1-4
times a day, depends on the patient's condition and weight.
[0029] In one embodiment, the pharmaceutical composition is
administrated subcutaneously to the subject. The dosage for
subcutaneous administration is generally 0.3-20, and preferably
0.3-3 mg/kg/day.
[0030] In one embodiment, the composition is applied topically onto
the affected area and rubbed into it. The composition is topically
applied at least 1 or 2 times a day, or 3 to 4 times per day,
depending on the medical issue and the disease pathology. In
general, the topical composition comprises about 0.01-20%, or
0.05-20%, or 0.1-20%, or 0.2-15%, 0.5-10, or 1-5% (w/w) of the
active compound. Typically 0.2-10 mL of the topical composition is
applied to the individual per dose. The active compound passes
through skin and is delivered to the site of discomfort.
[0031] Those of skill in the art will recognize that a wide variety
of delivery mechanisms are also suitable for the present
invention.
[0032] The present invention is useful in treating a mammal
subject, such as humans, horses, dogs and cats. The present
invention is particularly useful in treating humans.
[0033] The following examples further illustrate the present
invention. These examples are intended merely to be illustrative of
the present invention and are not to be construed as being
limiting.
EXAMPLES
[0034] All animal studies were conducted under protocols approved
by APLAC from Stanford University. Older and young animals on
C57BL6 background were purchased from Taconic. Older animals are
defined as those above 36 weeks of age, when physiological aging
process accelerates in mice, while young counterparts are 20 weeks
of age. Data are presented as mean SEM. Two-tailed Student's t-test
and two-way ANOVA were used for statistical analyses. A p value of
<0.05 is considered to be statistically significant.
Example 1. Ppargc1a Activator ZLN005 Suppresses Metabolic
Dysfunction in Microglia in Older Mice
[0035] Older animals were orally treated 3 times a week for 15
weeks with 0.5% methylcellulose (vehicle) or ZLN005 (Sigma) at 25
mg/kg in vehicle, starting at 37 weeks of age. Treated older
animals (n=6), non-treated older animals (n=6), and young animals
(20 weeks old, n=6) were sacrificed after drug treatment and their
brain tissues collected. PBS-perfused brain tissues of sacrificed
animals were digested with Collagenase I and processed for flow
cytometry (Ginhoux et al Science, 330:841-5, 2010). Brain microglia
were phenotyped with 2-NBDG
(2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose,
Invitrogen) and anti-Slc2a1 antibody (RnD) for flow cytometric
acquisition (LSRII, BD) and analysis (FlowJo).
[0036] The results are summarized in FIG. 1; Y-axis represents % of
microglia that either express glucose transporter Slc2a1 or have
taken up glucose fluorescent analog 2-NBDG. ANOVA was used for
statistical analysis. The results show that microglia in older mice
exhibited a glycolytic activation phenotype, evidenced by a
significant increase in Slc2a1 expression as well as 2-NBDG uptake
in vehicle treated older mice when compared with young mice
(p-values <0.01). The results also show that by administering
ZLN005 to older animals, Slc2a1 expression and glucose uptake in
microglia of these treated animals decreased, and thus their
metabolic dysfunctions were alleviated. (p-values <0.05).
Example 2. Ppargc1a Activator ZLN005 Inhibits Inflammatory Cytokine
Production in Microglia in Older Mice
[0037] Older animals were orally treated 3 times a week for 15
weeks with 0.5% methylcellulose (vehicle) or ZLN005 (Sigma) at 25
mg/kg in vehicle, starting at 37 weeks of age. Treated older
animals (n=6), non-treated older animals or aged animals (n=6), and
young animals (20 weeks old, n=6) were sacrificed after drug
treatment and their brain tissues were collected. PBS-perfused
brain tissues of sacrificed animals were digested with Collagenase
I and processed for flow cytometry (Ginhoux et al Science,
330:841-5, 2010). Brain microglia were phenotyped with
fluorochrome-labeled antibodies to CCL2 and TNF-.alpha. (Biolegend)
for flow cytometric acquisition (LSRII, BD) and analysis
(FlowJo).
[0038] The results are summarized in FIG. 2; Y-axis represents % of
microglia that express CCL2 or TNF-.alpha.. ANOVA was used for
statistical analysis. The results show that microglia in older mice
exhibited an inflammatory phenotype, evidenced by a significant
increase in CCL2 and TNF-.alpha. production in vehicle-treated
older mice when compared with young mice. The results also show
that by administering ZLN005 to older animals, CCL2 and TNF-.alpha.
production in microglia of these treated, older animals decreased
and thus neuroinflammation was suppressed (p-values <0.05 for
TNF-.alpha. and <0.01 for CCL2).
Example 3. Ppargc1a Activator ZLN005 Suppresses Systemic
Inflammation in Older Mice
[0039] TNF-.alpha. levels in serum of young mice, vehicle-treated
older mice, and ZLN-treated older mice (from Example 2) were
measured by ELISA to determine TNF-.alpha. levels which indicated
systemic inflammation. The results are summarized in FIG. 3; Y-axis
represents the concentration of TNF-.alpha. in serum. The results
show that by administering ZLN005 to older animals, TNF-.alpha.
levels in serum of these treated and older animals decreased and
thus systemic inflammation was suppressed when compared with
vehicle-treated older animal (p-value <0.05). Unpaired t-test
was used for statistical analysis.
Example 4. Ppargc1a Activator ZLN005 Alleviates Behavioral
Dysfunction in Older Mice
[0040] Animals were orally treated 3 times a week for 15 weeks with
0.5% methylcellulose (vehicle) or ZLN005 (Sigma) at 25 mg/kg in
vehicle, starting at 37 weeks of age. At 52 weeks of age, the older
animals were subjected to a marble burying assay, a behavioral test
of anxiety (Dekeyne A, Therapie, 60:477-84, 2005). Each animal was
given 12 marbles on top of bedding in individual cage and allowed
to run free in the cage for 30 minutes. After this period of time,
each buried marble was individually assigned a number based on its
degree of being buried (1=90-100% hidden, 0.75=60-90% hidden,
0.5=30-60% hidden, 0=below 30% hidden), and then the sum of the
numbers of 12 marbles buried by each mouse is calculated as the
marble burying score of the mouse. The higher score an animal had,
the higher anxiety index it exhibited. Results in FIG. 4 reveal
that older mice treated with vehicle had a higher average score
than those treated with ZLN005 (p-value <0.05), indicating that
ZLN005 reduced the anxiety phenotype and corrected behavioral
dysfunction in older mice (n=8-9 mice per group). Unpaired t-test
was used for statistical analysis.
[0041] The invention, and the manner and process of making and
using it, are now described in such full, clear, concise and exact
terms as to enable any person skilled in the art to which it
pertains, to make and use the same. It is to be understood that the
foregoing describes preferred embodiments of the present invention
and that modifications may be made therein without departing from
the scope of the present invention as set forth in the claims. To
particularly point out and distinctly claim the subject matter
regarded as invention, the following claims conclude the
specification.
* * * * *